Progressing Mechanobiology from a Simplified to a More Complex System: Development of Novel Platforms and Investigation of Actin Cytoskeletal Remodeling

Abstract

Compression is a common mechanical stimulus in our body that results from gravity and tissue growth. Inability for cells to maintain force homeostasis can lead to various diseases, and cells may experience an increase in compressive stress in diseases such as hypertension and cancer. Actin cytoskeleton provides structural and mechanical strength for cells to withstand external mechanical forces. It is also very sensitive to deformation and plays an important role in mechanosensing, due to the tensegrity nature of the cytoskeleton. Despite being an important component, actin remodeling and molecular signaling in response to compression have not been very well studied. From a molecular perspective, the actin network is regulated by actin accessory proteins into different structures and functions. At a cellular level, different actin networks are organized and regulated by various signaling pathways. At an intercellular level, cells are mechanically coupled that enable the transmission of force via actin networks between cells. In this dissertation, I investigated the sensing and transduction of compression stimulus in cells from molecular to intercellular scales, I developed new emerging platforms for the compression studies in in vitro reconstitution and single-cell system, and investigated actin remodeling and its related mechanisms under compression in 2D cell population. First, I developed an approach that combines purified actin cytoskeletal proteins or mammalian cell-free expression and ultra-thin double emulsion template for constructing a simplified model of a cell through in vitro reconstitution. This was used to study the mechanical properties of a specific and isolated cytoskeleton structure. We demonstrated the formation of bundled actin filaments inside the lipid vesicles. By using the mammalian cell-free expression system, we found that actin structures inside the system were precipitating with the droplet stabilizing surfactant polyvinyl alcohol, leading to a compensation of protein production and vesicle stability. Nevertheless, this approach provides a simplified yet insightful cell-like model for future cell mechanics investigation. I also developed a pneumatic-controlled, two-layered microfluidic platform for applying compression and aspiration to double emulsion droplets as a model cell. I further improved the microfluidic device to apply controlled compression to single cells that can be used to study the heterogeneity of mechanical properties and responses of cells. The device was designed through optimization by simulation and was characterized experimentally. Static and cyclic compressions were applied to single cells seeded inside the device. Finally, I investigated actin cytoskeletal remodeling, mechanosensing and mechanotransduction of epithelial cells under compression, in a 2D cell population context. I discovered the formation of actin protrusions at the apical surface of the cells under 1200 Pa compression. The actin protrusions were structurally similar to invadopodia but not functionally. Src signaling was found to be an important signaling pathway in these actin protrusions. This discovery opens up new direction of research and may explain why cells become more invasive under compression. This work could also shed light on the heterogeneity of cancer tissues and may inspire a new treatment paradigm. In this dissertation, I developed experimental platforms and studied the remodeling of actin networks under compression using in vitro reconstitution, single-cell and cell population systems, taking advantages of each experimental system. The platforms developed in this dissertation provide novel techniques for investigating actin cytoskeleton response of cell under compression, and the discovery of actin protrusions reported in this dissertation reveals a new cellular response under high compression.